RESEARCH ARTICLE 3423

Development 140, 3423-3434 (2013) doi:10.1242/dev.095083 © 2013. Published by The Company of Biologists Ltd Essential role for ligand-dependent feedback antagonism of vertebrate hedgehog signaling by PTCH1, PTCH2 and HHIP1 during neural patterning Alexander M. Holtz1,2,3, Kevin A. Peterson4,*, Yuichi Nishi4,*, Steves Morin5, Jane Y. Song1, Frédéric Charron5,6,7, Andrew P. McMahon4,8,9,*,‡ and Benjamin L. Allen1,‡

SUMMARY Hedgehog (HH) signaling is essential for vertebrate and invertebrate embryogenesis. In Drosophila, feedback upregulation of the HH receptor Patched (PTC; PTCH in vertebrates), is required to restrict HH signaling during development. By contrast, PTCH1 upregulation is dispensable for early HH-dependent patterning in mice. Unique to vertebrates are two additional HH-binding antagonists that are induced by HH signaling, HHIP1 and the PTCH1 homologue PTCH2. Although HHIP1 functions semi-redundantly with PTCH1 to restrict HH signaling in the developing nervous system, a role for PTCH2 remains unresolved. Data presented here define a novel role for PTCH2 as a ciliary localized HH pathway antagonist. While PTCH2 is dispensable for normal ventral neural patterning, combined removal of PTCH2- and PTCH1-feedback antagonism produces a significant expansion of HH-dependent ventral neural progenitors. Strikingly, complete loss of PTCH2-, HHIP1- and PTCH1-feedback inhibition results in ectopic specification of ventral cell fates throughout the neural tube, reflecting constitutive HH pathway activation. Overall, these data reveal an essential role for ligand- dependent feedback inhibition of vertebrate HH signaling governed collectively by PTCH1, PTCH2 and HHIP1.

KEY WORDS: Neural tube, Hedgehog, Negative feedback

INTRODUCTION governed by the canonical receptor Patched (PTC; PTCH in During embryogenesis, complex gene expression patterns arise vertebrates) (Chen and Struhl, 1996). Seminal studies in Drosophila within fields of initially equivalent cells to form the tissues that identified two distinct forms of PTC-mediated antagonism (Chen comprise a fully developed organism (Perrimon and McMahon, and Struhl, 1996). In the absence of ligand, PTC inhibits the activity 1999). A small number of conserved signaling pathways act via of Smoothened (SMO) (Ingham et al., 2000), a key effector of the secreted ligands to establish these embryonic patterns, producing pathway, in a process termed ligand-independent antagonism (LIA) distinct cellular responses in a concentration- and time-dependent (Jeong and McMahon, 2005). Ligand binding to PTC relieves SMO manner (Freeman, 2000; Kutejova et al., 2009; Ulloa and Briscoe, inhibition and culminates in modulation of HH target genes, 2007). These pathways require precise spatial and temporal control including ptc itself. Consequently, PTC is highly upregulated near of ligand production and distribution to preserve the requisite the source of HH production to bind and sequester ligand and limit diversity of cellular responses and to limit signaling within the the level and the range of signaling within a responding tissue appropriate domains. Thus, feedback antagonism of secreted ligands (Hooper and Scott, 1989; Nakano et al., 1989). This negative plays a crucial role in regulating the level and spatial distribution of feedback by PTC at the level of ligand is known as ligand- signaling within a target field (Freeman, 2000; Perrimon and dependent antagonism (LDA) (Jeong and McMahon, 2005). McMahon, 1999). Evidence from Drosophila suggests that PTC upregulation is Hedgehog (HH) proteins are secreted molecules that play crucial dispensable for SMO inhibition (LIA), but is required to sequester roles in both vertebrate and invertebrate development (McMahon et HH ligand and prevent pathway activation in cells more distal to al., 2003). Negative feedback at the level of the HH ligand is the HH source (LDA) (Chen and Struhl, 1996). Feedback upregulation of the vertebrate PTCH1 receptor is conserved in

1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, mammals (Goodrich et al., 1996); however, similar experiments that MI 48109, USA. 2Medical Scientist Training Program, University of Michigan, Ann abrogate PTCH1-feedback upregulation in mice do not dramatically Arbor, MI 48109, USA. 3Cellular and Molecular Biology Program, University of alter HH signaling during early embryogenesis (Jeong and 4 Michigan, Ann Arbor, MI 48109, USA. Department of Stem Cell and Regenerative McMahon, 2005; Milenkovic et al., 1999). In this model, tonal Biology, Harvard University, Cambridge, MA 02138, USA. 5Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montreal (IRCM), levels of PTCH1 are produced from a transgene using the Montreal, QC H2W 1R7, Canada. 6Department of Medicine, University of Montreal, metallothionein promoter (MT-Ptch1) (Milenkovic et al., 1999). In 7 Montreal, QC H3T 1J4, Canada. Faculty of Medicine, McGill University, Montreal, MT-Ptch1;Ptch1−/− embryos, basal levels of PTCH1 are sufficient QC H3G 1YG, Canada. 8Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA. 9Department of Molecular and Cellular Biology, Harvard University, for LIA, but surprisingly, given the Drosophila studies, these Cambridge, MA 02138, USA. embryos display a largely normal body plan at E10.5, with relatively minor disturbances of HH-dependent patterning (Jeong and *Present address: Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad-CIRM Center for Regenerative Medicine and Stem Cell Research, McMahon, 2005; Milenkovic et al., 1999). University of Southern California Keck School of Medicine, Los Angeles, CA 90089, In contrast to Drosophila, vertebrates possess two additional cell USA surface HH-binding proteins that are induced by HH signaling: ‡Authors for correspondence ([email protected]; [email protected]) hedgehog interacting protein 1 (HHIP1; HHIP – Mouse Genome

Accepted 8 June 2013 Informatics) (Chuang and McMahon, 1999), a membrane-anchored DEVELOPMENT 3424 RESEARCH ARTICLE Development 140 (16) glycoprotein, and patched 2 (PTCH2; Motoyama et al., 1998b), a globin insulator. Transient transgenics were generated via pronuclear structural homolog of PTCH1 that arose from a gene duplication injection and collected at E10.5. PCR genotyping and X-gal staining were event. HHIP1 acts partially redundantly with PTCH1 to antagonize performed as previously described (Vokes et al., 2007). HH signaling in the developing mouse central nervous system Chick in ovo neural tube electroporations (CNS); embryos lacking both HHIP1- and PTCH1-feedback Electroporations were performed as previously described (Allen et al., inhibition generate cell fates within the normal HH signaling 2011). In brief, DNA (1.0 μg/μl) was injected into the neural tubes of domain that are more ventral than expected, and HH responses Hamburger-Hamilton stage 10-12 chicken embryos with 50 ng/μl Fast extend into dorsal regions that do not normally exhibit active Green. Embryos were dissected after 48 hours and fixed in 4% PFA for signaling (Jeong and McMahon, 2005). By comparison, mutant immunofluorescent analysis. embryos such as Ptch1−/−, where LIA is removed, and Sufu−/−, Immunofluorescence where the pathway is activated downstream of ligand, have an even Immunofluorescence was performed essentially as previously described more ventralized phenotype with a significant extent of the neural (Allen et al., 2011). Neural patterning analysis was performed at the tube adopting a Sonic HH (SHH)-secreting floorplate fate (Cooper forelimb level in E9.5 and E10.5 embryos. The following antibodies were et al., 2005; Goodrich et al., 1997). The differences in the severity used: mouse IgG1 anti-NKX6.1 [1:20, Developmental Studies Hybridoma of these patterning defects suggest that other mechanisms limit Bank (DSHB)], mouse IgG2a anti-PAX3 (1:20, DSHB), rabbit IgG anti- ligand-dependent vertebrate HH pathway activity. DBX1 (1:1000, gift from Dr Yasushi Nagakawa, University of Minnesota, The contribution of PTCH2 in feedback antagonism during CNS Minneapolis, MN, USA), mouse IgG1 anti-FOXA2 (1:20, DSHB), rabbit patterning has not been addressed. Mouse PTCH1 and PTCH2 share IgG anti-FOXA2 (1:500, Cell Signaling), mouse IgG2b anti-NKX2.2 (1:20, DSHB), rabbit IgG anti-OLIG2 (1:1000, Millipore), mouse IgG1 anti-SHH 56% amino acid identity; a key difference is that PTCH2 is more (1:20, DSHB), rabbit IgG anti-cleaved caspase 3 (1:200, Cell Signaling), stable than PTCH1 due to a truncated C-terminal region (Kawamura rabbit IgG anti-phospho-histone H3 (1:1000, Millipore), mouse IgG1 anti- et al., 2008). Although PTCH2 antagonizes HH signaling in cell- MNR2 (1:20, DSHB), mouse IgG2b anti-ISL1 (1:20, DSHB), mouse IgG2a based assays (Rahnama et al., 2004), Ptch2−/− mice are viable and anti-EVX1 (1:20, DSHB) and mouse IgG1 anti-EN1 (1:20, DSHB). Nuclei fertile, whereas Ptch1−/− mice die at E9.5 with ectopic HH signaling were visualized using DAPI (1:30,000, Molecular Probes). Alexa 488, 555 throughout the embryo (Goodrich et al., 1997; Nieuwenhuis et al., and 633 secondary antibodies (1:500, Molecular Probes) were visualized 2006). Ptch2−/− embryos do, however, exhibit subtle changes in on a Leica upright SP5X confocal microscope. gene expression that are consistent with increased HH pathway Cellular localization of HH pathway components activation, including a slight expansion of Ptch1 and Gli1 NIH/3T3 fibroblasts were plated at 150,000 cells/well on coverslips and expression in the developing limb bud and the embryonic hair transfected 16-24 hours later. Six hours post-transfection, cells were placed follicle (Nieuwenhuis et al., 2006). These transcriptional changes into low-serum (0.5%) media and fixed 48 hours later in 4% PFA for ultimately resolve to produce normally patterned HH-responsive immunofluorescent analysis. tissues, although aged male Ptch2+/− and Ptch2−/− mice develop Luciferase assays epidermal hyperplasia and alopecia (Nieuwenhuis et al., 2006). That Luciferase assays were adapted from a previously published protocol PTCH1 action may mask PTCH2 activity is a reasonable (Nybakken et al., 2005). Mouse NIH/3T3 fibroblasts were plated at 25,000 hypothesis, especially given the observation that the loss of Ptch2 cells/well on gelatinized 24-well plates and transfected 16-24 hours later enhances tumorigenesis in a Ptch1+/− background (Lee et al., 2006). with 150 ng of a ptc∆136-GL3 luciferase reporter (Chen et al., 1999; Here, we demonstrate that PTCH2 is a crucial component of LDA Nybakken et al., 2005), 50 ng of pSV-β-galactosidase (Promega) and 100 in the developing neural tube. Although embryos lacking PTCH2 ng of control (pCIG) or experimental constructs using Lipofectamine 2000 alone or in combination with HHIP1 display normal neural patterning, (Invitrogen). After 48 hours, cells were placed in low serum (0.5%) media combined loss of PTCH2- and PTCH1-feedback inhibition results in with 25 μl of conditioned media from control (pCDNA3) or NSHH a significant expansion of SHH-dependent ventral cell populations. In transfected (NShh-pCDNA3) COS7 cells. Luciferase (Luciferase Assay System Kit, Promega) and β-galactosidase (BetaFluor β-galactosidase addition, complete loss of PTCH2-, HHIP1- and PTCH1-feedback Assay Kit, Novagen) activity were measured after 48 hours. Luciferase inhibition results in a neural tube composed entirely of ventral cell values were normalized to β-galactosidase activity and expressed as fold −/− −/− populations, similar to Ptch1 and Sufu embryos. Overall, these induction relative to control treated cells. data demonstrate an essential role for negative feedback at the level Signaling assays in Ptch1−/− MEFs (a gift from Dr M. P. Scott, Stanford of HH ligand during vertebrate development, and reveal a collective University, CA, USA) were performed as described with the following requirement for PTCH1, PTCH2 and HHIP1 in ligand-dependent modifications. Ptch1−/− MEFs were plated at 50,000 cells per well and feedback inhibition. secreted placental alkaline phosphatase was used as a transfection control (Alkaline Phosphatase Yellow pNPP Liquid Substrate for ELISA, Sigma- MATERIALS AND METHODS Aldrich). Mice Immunoprecipitation Ptch2 mice were generated and provided by Curis. The absence of Ptch2 COS7 cells were plated at 1×106 cells/100 mm dish and transfected the next mRNA was confirmed by expression analysis in the testes, the highest site day with expression plasmids for the indicated proteins. of Ptch2 expression (Carpenter et al., 1998). Hhip1 (Chuang et al., 2003), Immunoprecipitations and western blot analyses were performed as Ptch1 (Goodrich et al., 1997) and MtPtch1 (Milenkovic et al., 1999) mice previously described (Okada et al., 2006). In brief, PTCH::HA proteins were have all been previously described. For timed pregnant analyses, noon of the immunoprecipitated using a mouse anti-HA antibody (SIGMA H3663). day on which a vaginal plug was detected was considered E0.5. Precise Western blot analyses were then performed using mouse anti-HA, rabbit embryo staging was achieved by assessment of somite number at the time anti-GFP (Molecular Probes A11122), goat anti-Gas1 (R&D AF2644) and of dissection. For each analysis, a minimum of three embryos were analyzed mouse anti-Actin (SIGMA A5441) to reveal the input and IP levels. and representative images are shown. For transgenic analysis of the Ptch2 enhancer, the Ptch2 enhancer region In situ hybridization (chr4:116,768,296-116,768,754) was PCR amplified from C57Bl/6J In situ hybridization was performed essentially as described previously genomic DNA, sequence verified and cloned upstream of a modified (Wilkinson, 1992) using digoxigenin-labeled probes on 20 μm sections

Hsp68-lacZ reporter construct containing a single copy of the chicken β- collected at the forelimb level of E9.5 and E10.5 embryos. DEVELOPMENT Inhibition of HH signaling RESEARCH ARTICLE 3425

RESULTS conserved promoter proximal GBR (Ptch2(−0.5kb); Fig. 1A); Ptch2 is a direct HH target that antagonizes SHH- however, the regulatory potential of this region remains mediated pathway activation in vivo unexplored. To determine the enhancer activity associated with Genomic characterization of GLI1- and GLI3-binding profiles Ptch2(−0.5kb), we isolated a highly conserved 459 bp region and highlight HH pathway components, including Ptch1, Ptch2 and assayed reporter expression in transient transgenic mouse embryos Hhip1, as direct transcriptional targets of HH signaling (Peterson at E10.5 (Fig. 1B). The Ptch2(−0.5kb) enhancer displays a ventral et al., 2012; Vokes et al., 2007; Vokes et al., 2008). Previously, a distribution throughout the entire CNS, consistent with Ptch2 as a promoter proximal GLI1-binding region (GBR) associated with direct readout of SHH signaling (Fig. 1C,D) and in line with Ptch1 recapitulated the majority of Ptch1 expression, including in previous reports of Ptch2 expression in the neural tube (Motoyama the CNS (Vokes et al., 2007). Similarly, Ptch2 possesses a et al., 1998a).

Fig. 1. PTCH2 is a direct transcriptional target that antagonizes HH signaling in NIH/3T3 cells and in the developing chick neural tube. (A) Ptch2 regulatory landscape highlighting two discrete GLI1 binding events positioned at −0.5 kb and +2.2 kb relative to TSS. (B) Higher magnification view of −0.5 kb region assayed for enhancer activity (blue bar). Computationally predicted GLI-binding sites (GBS) are shown in black (nonconserved) and red (conserved). Multi-species conservation (cons.) is shown below. (C) Transient transgenic analysis of Ptch2(−0.5 kb) regulatory region shows neural- specific activity at E10.5. The number of embryos expressing the transgene out of total transgenic positives is shown in the upper right-hand corner. (D) Transverse section taken from region indicated in C (black bar) shows reporter activity restricted to the ventral neural tube. (E) HH-responsive luciferase reporter activity measured in NIH/3T3 fibroblasts stimulated with either control media (white bars) or NSHH-conditioned media (grey bars), and co-transfected with the indicated constructs. Each condition was performed in triplicate and data are represented as mean±s.e.m., P-values measured by two-tailed Student’s t-test. (F-U) Hamburger-Hamilton stage 19-22 chick neural tubes electroporated with pCIG (F-I), Ptch1::HA-pCIG (J-M), Ptch2::HA-pCIG (N-Q) and Ptch1ΔL2::HA-pCIG (R-U) sectioned at the wing level and stained with antibodies raised against NKX6.1 (red, F,G,J,K,N,O,R,S) or PAX7 (red, H,I,L,M,P,Q,T,U). Nuclear EGFP expression (G,I,K,M,O,Q,S,U) labels electroporated cells. Arrows indicate repression of NKX6.1 expression (J,K,N,O,R,S) or ectopic expression of PAX7 (L,M,P,Q,T,U). Arrowheads indicate ventrally located electroporated cells that maintain NKX6.1 expression

(J,K,N,O) or lack ectopic PAX7 expression (L,M,P,Q). Scale bar: 50 μm in F-U. DEVELOPMENT 3426 RESEARCH ARTICLE Development 140 (16)

To examine PTCH2-mediated antagonism of HH signaling, and exhibit no overt defects in ventral neural patterning at E10.5 to compare PTCH2 directly with other cell surface HH pathway (Fig. 2B,G; supplementary material Fig. S1). antagonists, we expressed PTCH2, PTCH1 and HHIP1 in HH- To address possible redundancy between PTCH2 and HHIP1 responsive NIH/3T3 fibroblasts. Cells transfected with a HH- functions, we analyzed neural patterning in Ptch2;Hhip1 double responsive luciferase reporter (Chen et al., 1999) and treated with mutant embryos at E10.5 (Fig. 2); however, Ptch2−/−;Hhip1−/− SHH show robust induction of luciferase activity compared with embryos are grossly normal and exhibit no defects in ventral neural untreated cells (Fig. 1E). Consistent with previous reports, patterning at this stage (Fig. 2E,J; supplementary material Fig. S2C). expression of PTCH1, HHIP1 or PTCH2 inhibits NSHH-mediated pathway activation (Fig. 1E) (Rahnama et al., 2004). Expansion of ventral neural progenitors in To extend these results, we used chick in ovo electroporations to embryos lacking PTCH1- and PTCH2-feedback determine whether PTCH2 could antagonize SHH-dependent ventral antagonism neural patterning. During nervous system development, a gradient of Given the previously identified redundancy between PTCH1 and SHH directly induces (class II genes Nkx6.1, Nkx2.2, FoxA2, etc.) or HHIP1, we reasoned that PTCH1-feedback inhibition is sufficient indirectly represses (class I genes Pax7, Pax3, etc.) expression of a to antagonize SHH signaling in Ptch2;Hhip1 double mutants. Thus, series of transcriptional regulators in a concentration-dependent to uncover a role for PTCH2, we used an MT-Ptch1 transgene that manner (Dessaud et al., 2008). The combinatorial activity of these produces sufficient levels of PTCH1 for LIA of SMO (Milenkovic transcriptional determinants along the dorsal-ventral (DV) axis et al., 1999) in order to compare the phenotypes of MT- specifies unique neural progenitor domains that generate distinct Ptch1;Ptch1−/− embryos that lack PTCH1-mediated LDA with MT- classes of mature neurons (Briscoe et al., 2000). These targets Ptch1;Ptch1−/−;Ptch2−/− embryos, which are incapable of both quantitatively readout HH pathway activity in vivo. PTCH1 and PTCH2-dependent LDA. Ectopic expression of EGFP (pCIG) in the chick neural tube does As previously reported, MT-Ptch1;Ptch1−/− embryos display a not affect neural patterning (Fig. 1F-I) based on expression of grossly normal body plan at E10.5 (supplementary material Fig. NKX6.1 (class II target) and PAX7 (class I target). By contrast, S2D) (Jeong and McMahon, 2005; Milenkovic et al., 1999), overexpression of either PTCH1 or PTCH2 represses NKX6.1 although a subtle expansion of ventral cell identities is detected (Fig. 1J,K,N,O, arrows) and de-represses PAX7 expression in the when compared with wild-type embryos at E10.5 (Fig. 3A,B,E,F). ventral neural tube (Fig. 1L,M,P,Q, arrows), consistent with PTCH1 This is in stark contrast to analogous experiments performed in and PTCH2 acting as HH pathway antagonists. Importantly, Drosophila, where removal of PTC-feedback inhibition completely NKX6.1 is maintained and PAX7 remains repressed in more ventral abrogates receptor-mediated feedback antagonism (Chen and cells electroporated with either PTCH1 or PTCH2 (Fig. 1J-Q, Struhl, 1996). Intriguingly, MT-Ptch1;Ptch1−/−;Ptch2−/− embryos arrowheads), suggesting that patched-mediated antagonism can be exhibit midbrain and hindbrain exencephaly (supplementary overcome by higher ligand concentrations. By contrast, a ligand- material Fig. S2E) – similar to mutants lacking GLI3 repressor insensitive PTCH1 (PTCH1ΔL2) that acts as a constitutive SMO activity (Hui and Joyner, 1993) and consistent with overactive HH antagonist (Briscoe et al., 2001) inhibits NKX6.1 and enables PAX7 pathway activity. Compared with MT-Ptch1;Ptch1−/− embryos, MT- expression in a position-independent manner (Fig. 1R-U). Taken Ptch1;Ptch1−/−;Ptch2−/− embryos also exhibit significant expansion together, these data are consistent with PTCH2 acting as a HH of SHH-dependent NKX6.1 (Fig. 3D), FOXA2, NKX2.2 and pathway antagonist. OLIG2 (Fig. 3H) expression at E10.5, which is indicative of an increased range of HH signaling in the absence of PTCH2. In Ptch2−/− and Ptch2−/−;Hhip1−/− embryos display particular, NKX2.2+ cells, which require a high threshold for normal ventral neural patterning induction (Ericson et al., 1997), are dorsally extended in MT- To determine the endogenous actions of PTCH2, we analyzed SHH- Ptch1;Ptch1−/−;Ptch2−/− embryos (Fig. 3H, arrows, see inset). The dependent ventral neural patterning in Ptch2−/− embryos. Consistent ventral expansion is accompanied by retraction of the dorsal PAX3+ with other published alleles, Ptch2−/− mice are viable and fertile domain in MT-Ptch1;Ptch1−/−;Ptch2−/− embryos (Fig. 3D). (Nieuwenhuis et al., 2006) and display a grossly normal body plan Quantitation demonstrates a significant increase in FOXA2+ floor- at E10.5 (supplementary material Fig. S2A,B). As reported for plate cells (Fig. 3I), NKX2.2+ v3 interneuron progenitors (Fig. 3J) Hhip1 mutants (Jeong and McMahon, 2005), Ptch2−/− embryos and the proportion of the neural tube that is NKX6.1+ in

Fig. 2. Normal SHH-mediated ventral neural patterning in E10.5 Ptch2−/−;Hhip1−/− mouse embryos. (A-J) Immunofluorescent analysis of neural patterning in E10.5 mouse forelimb sections detects expression of NKX6.1, DBX1, PAX3 (red, green and magenta, respectively; A-E), FOXA2, NKX2.2 and OLIG2 (red, green and magenta, respectively; F-J) in wild- type (A,F), Ptch2−/− (B,G), Hhip1−/− (C,H), Ptch2−/−;Hhip1+/− (D,I) and Ptch2−/−,Hhip1−/− (E,J) embryos. Scale bars: 50 μm. DEVELOPMENT Inhibition of HH signaling RESEARCH ARTICLE 3427

Fig. 3. Expansion of SHH-dependent ventral progenitor domains in E10.5 mouse embryos lacking both PTCH2- and PTCH1-feedback antagonism. (A-H) Neural patterning analysis in E10.5 forelimb sections using antibodies against NKX6.1, DBX1, PAX3 (red, green and magenta, respectively; A-D), FOXA2, NKX2.2 and OLIG2 (red, green and magenta, respectively; E-H) in wild type (A,E), MT-Ptch1;Ptch1−/− (B,F), MtPtch1;Ptch1−/−;Ptch2+/− (C,G) and MT-Ptch1;Ptch1−/−;Ptch2−/− (D,H) embryos. Insets show NKX2.2 channel alone (E-H). Arrows indicate dorsal expansion of NKX2.2+ cells in MT-Ptch1;Ptch1−/−;Ptch2−/− embryos (H). (I-K) Quantitation of FOXA2+ cell number (I), NKX2.2+ cell number (J) and NKX6.1 domain size as a % of total DV neural tube length (K). Data are represented as mean±s.e.m. calculated from at least three embryos per genotype. P-values are determined by two-tailed Student’s t-test. Scale bars: 50 μm.

MT-Ptch1;Ptch1−/−;Ptch2−/− embryos compared with MT- length of the DV axis and only a small number of PAX3+ cells Ptch1;Ptch1−/− animals (Fig. 3K). remain (Goodrich et al., 1997). This disparity could be explained These results, in combination with previous studies (Jeong and by the residual functions of HHIP1 or PTCH2 in MT- McMahon, 2005), demonstrate that PTCH2 and HHIP1 functionally Ptch1;Ptch1−/−;Ptch2−/− or MT-Ptch1;Ptch1−/−;Hhip1−/− embryos, compensate for the absence of PTCH1-feedback inhibition during respectively. ventral neural patterning. To determine whether transcriptional To test this, we reduced the gene dose of Hhip1 in MT- upregulation of Ptch2 or Hhip1 occurs in the absence of PTCH1- Ptch1;Ptch1−/−;Ptch2−/− embryos. Consistent with this view, MT- mediated LDA, we examined Ptch2 and Hhip1 expression patterns Ptch1;Ptch1−/−;Ptch2−/−;Hhip1+/− embryos display a more severe in the neural tube of MT-Ptch1;Ptch1−/− embryos using RNA in situ expansion of ventral cell populations than MT- hybridization (supplementary material Fig. S3). Both Hhip1 and Ptch1;Ptch1−/−;Ptch2−/− embryos (Fig. 4B,C,F,G). Additionally, Ptch2 are transcriptionally upregulated in the ventral neural tube of MT-Ptch1;Ptch1−/−;Hhip1−/−;Ptch2+/− embryos exhibit a further MT-Ptch1;Ptch1−/− compared with wild-type embryos at E9.5 and expansion of ventral cell identities compared with MT- E10.5 (supplementary material Fig. S3). We also observe significant Ptch1;Ptch1−/−;Hhip1−/− embryos (Fig. 4J,K,N,O). Thus, both upregulation of Hhip1 transcripts in the paraxial mesoderm in HHIP1 and PTCH2 play significant roles when PTCH1/PTCH2 or embryos lacking PTCH1-feedback inhibition (supplementary PTCH1/HHIP1 feedback responses are removed, respectively. Of material Fig. S3M-P). note, there is significant variability in the degree of patterning Together, the data suggest that PTCH1, PTCH2 and HHIP1 all defects in these embryos, which likely reflects the large effects from contribute to LDA of SHH signaling. Furthermore, when PTCH2 or fluctuations in near-threshold levels of dorsal SHH signals HHIP1 is absent, the normal patterning response is dependent on (Fig. 4K,O, insets). Interestingly, we also observed significant PTCH1-mediated LDA. mixing among different cell populations, indicating that LDA is essential to generate discrete boundaries between progenitor Severe neural tube ventralization in E10.5 domains (Fig. 4F,G, arrows). embryos lacking combined PTCH1-, PTCH2- and These data support the notion that PTCH1, PTCH2 and HHIP1 HHIP1-feedback antagonism together comprise a feedback network of cell surface HH In both MT-Ptch1;Ptch1−/−;Ptch2−/− and MT-Ptch1;Ptch1−/−; antagonists. To test this hypothesis, we generated embryos that Hhip1−/− embryos, a persistent PAX3+; NKX6.1– dorsal domain completely lack cell surface feedback antagonism (MT- suggests that SHH signaling is largely absent from the dorsal neural Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/−). tube (Fig. 3, Fig. 4B,J). The cells in this region are HH responsive, Grossly, triple mutant embryos display severe exencephaly −/−

as evident from Ptch1 embryos where NKX6.1 extends the throughout most of the anterior-posterior axis, CNS overgrowth, DEVELOPMENT 3428 RESEARCH ARTICLE Development 140 (16)

Fig. 4. Severe neural tube ventralization in E10.5 MT- Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− embryos. (A-U) Antibody detection of NKX6.1, DBX1, PAX3 (red, green and magenta, respectively; A-D,I-L), FOXA2, NKX2.2, OLIG2 (red, green and magenta, respectively; E-H,M-P), SHH (5E1) and FOXA2 (green and red, respectively; Q-U) in E10.5 forelimb sections from wild type (A,E,I,M,Q), MT-Ptch1;Ptch1−/− (R), MT-Ptch1;Ptch1−/−;Ptch2−/− (B,F,S), MT-Ptch1;Ptch1−/−;Hhip1−/− (J,N,T), MT-Ptch1;Ptch1−/−;Ptch2−/−;Hhip1+/− (C,G), MT- Ptch1;Ptch1−/−;Hhip1−/−;Ptch2+/− (K,O) and MT- Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− (D,H,L,P,U) embryos. Arrows indicate NKX2.2+ cells within the OLIG2 domain (F) and OLIG2+ cells in the NKX2.2 domain (G). Insets are representative of less severe phenotypes that are observed in MT-Ptch1;Ptch1−/−;Hhip1−/−;Ptch2+/− embryos (K,O). Scale bars: 50 μm.

craniofacial abnormalities and enlarged somites (supplementary demonstrate a collective requirement for PTCH2-, HHIP1- and material Fig. S2G). Remarkably, MT-Ptch1;Ptch1−/−;Ptch2−/−; PTCH1-feedback inhibition to restrict HH signaling in order to Hhip1−/− embryos exhibit neural patterning defects comparable ensure the appropriate diversity of both ventral and dorsal neural with those described in Ptch1−/− embryos (Fig. 4D,H,L,P) progenitor types. (Goodrich et al., 1997): a small dorsal midline cluster of PAX3+ Beyond the severe neural patterning defects observed in the cells, NKX6.1 expression along the entire DV axis (Fig. 4D,L), embryos, we also detected significant deficits in the size and OLIG2+ motoneuron progenitors confined to the dorsal neural tube, cellularity of MT-Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− neural tubes, and NKX2.2+ v3 progenitors extending to the dorsal limits of the as well as aberrant neuro-epithelial outgrowths (supplementary neuraxis (Fig. 4H,P; supplementary material Fig. S4). material Fig. S6A-E, arrows) and cells budding off of the epithelium FOXA2 is crucial for induction of SHH at the ventral midline into the lumen (supplementary material Fig. S6F-J, arrowheads). and its activation there requires the highest level of HH signaling These morphological defects are accompanied by reduced (Ribes et al., 2010; Roelink et al., 1995). In MT- proliferation and increased apoptosis, as assessed by phospho- Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− embryos, FOXA2 production histone H3 (PH3) and cleaved caspase 3 (CC3) staining, extends throughout the DV axis with high levels of expression respectively, in triple mutants (supplementary material Fig. S7). ventrally and low levels dorsally (Fig. 4H,P; supplementary material Notably, apoptotic cells are most prominent in the paraxial Fig. S4), resulting in a dramatically enlarged SHH-producing mesoderm surrounding the neural tube (supplementary material Fig. floorplate (Fig. 4Q-U). However, persistent NKX2.2 expression in S7) and the distal extent of apoptotic mesodermal cells from the these cells demonstrates incomplete floorplate maturation notochord increases with the severity of the LDA mutations, (Fig. 4H,P; supplementary material Fig. S4). suggesting that the cell death is dependent on SHH ligand. As an expected outcome of progenitor misspecification, we also Additionally, the loss of paraxial mesoderm could contribute to the observed a severe reduction in post-mitotic descendants of specific lack of mature neurons (supplementary material Fig. S5) owing to progenitor classes in the absence of all LDA, including motoneurons compromised retinoic acid production from the somites, which is (ISL1 and MNR2), V1 interneurons (EN1) and V0 interneurons required for neuronal differentiation in the neural tube (Diez del

(EVX1; supplementary material Fig. S5). Overall, these data Corral et al., 2003; Novitch et al., 2003; Sockanathan et al., 2003). DEVELOPMENT Inhibition of HH signaling RESEARCH ARTICLE 3429

Severe neural tube ventralization in E8.5 MT- progenitors to limit HH signaling at the onset of ventral neural Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− embryos is patterning. independent of floorplate-derived SHH The extended SHH domain in MT-Ptch1;Ptch1−/−;Ptch2−/−; PTCH2 is a ciliary-localized SMO antagonist Hhip1−/− embryos raises the possibility that the observed patterning Although the ectopic signaling observed in MT- defects are secondary to enhanced SHH ligand production rather Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− embryos is likely ligand than due to a direct deficiency of LDA. To resolve these conflicting dependent, loss of inhibition downstream of ligand could also interpretations, we examined neural patterning at E8.5, where SHH- contribute to this phenotype. To address this, we tested whether dependent ventral patterning derives solely from notochord- PTCH1, PTCH2 or HHIP1 could antagonize signaling downstream expressed ligand. Although FOXA2 expression begins around the of SMO. In NIH/3T3 cells, co-transfection of PTCH1, PTCH2 or 8-somite stage, floor-plate SHH expression does not initiate until HHIP1 with constitutively active SMOM2 (Xie et al., 1998) does the 16-somite stage (Jeong and McMahon, 2005). Thus, patterning not reduce HH pathway activity compared with cells transfected defects prior to this time reflect direct readouts of the loss of LDA with SMOM2 alone (Fig. 6A); thus, these cell surface molecules uncomplicated by ectopic SHH from an expanded floorplate. act upstream of SMO. In Ptch2−/−;Hhip1−/− embryos at E8.5, neural patterning is We next explored whether PTCH1, PTCH2 and HHIP1 can normal, as indicated by the dorsal restriction of PAX3 and by directly antagonize SMO activity (LIA). To achieve this, we NKX6.1 expression in the ventral neural tube (Fig. 5A,F). In employed mouse embryonic fibroblasts (MEFs) isolated from addition, FOXA2 expression initiates in the ventral midline with Ptch1−/− embryos that lack LIA and exhibit high levels of HH SHH synthesis limited to the notochord (Fig. 5K). As expected, MT- signaling. Although PTCH1 can directly inhibit SMO, there are Ptch1;Ptch1−/− embryos exhibit a slight expansion of NKX6.1 and conflicting reports concerning LIA by PTCH2 (Nieuwenhuis et al., FOXA2 compared with Ptch2−/−;Hhip1−/− embryos (Fig. 5B,G,L). 2006; Rahnama et al., 2004). As previously reported, expression of However, MT-Ptch1;Ptch1−/−;Ptch2−/− embryos demonstrate a PTCH1 in Ptch1−/− MEFs causes robust inhibition of HH- dramatic expansion of NKX6.1 and FOXA2 expression compared responsive luciferase reporter activity even at low concentrations with MT-Ptch1;Ptch1−/− embryos (Fig. 5C,H,M) at E8.5. of transfected DNA (Fig. 6B) (Taipale et al., 2002). Although Interestingly, the magnitude of this early difference is more marked PTCH1 and PTCH2 function equivalently at high DNA than at E10.5, with FOXA2 expression at the dorsalmost extent of concentrations, PTCH2 displays significantly reduced activity at the neural tube in E8.5 MT-Ptch1;Ptch1−/−;Ptch2−/− and MT- lower concentrations, even though PTCH2 protein is highly stable Ptch1;Ptch1−/−;Ptch2−/−;Hhip1+/− embryos (Fig. 5M,N, arrows), compared with PTCH1 (Fig. 6B; Kawamura et al., 2008). By a phenotype never observed at E10.5. In some instances, we also contrast, HHIP1 is unable to inhibit SMO at any DNA concentration observed reduced PAX3 (Fig. 5H,I) and induction of FOXA2 tested (Fig. 6B). Overall, these results suggest that PTCH2 is (Fig. 5M,N) in the somites of LDA mutants at E8.5, suggesting that capable of LIA of SMO but that PTCH2 activity is weaker than that ligand-mediated feedback antagonism also functions to restrain HH of PTCH1. signaling in other HH-responsive tissues. Finally, MT- PTCH1 and PTCH2 are structurally related to the RND permease Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− embryos exhibit severe neural superfamily, which consists of 12-pass transmsembrane proteins tube ventralization that is nearly indistinguishable from Ptch1−/− that function by proton-antiport to efflux small molecules across embryos at E8.5 (Fig. 5J,O). In each instance, immunostaining for lipid bilayers (Tseng et al., 1999). This transporter activity is SHH confirmed that the patterning defects arise solely from dependent on a conserved RND domain, and missense mutations notochord-derived ligand (Fig. 5K-O). Collectively, these results within the PTCH1 RND motif result in impaired LIA, consistent are consistent with a direct requirement for LDA in neural with PTCH1 functioning catalytically as an RND transporter (Tseng

Fig. 5. Expansion of ventral progenitor domains occurs prior to floorplate expression of SHH in E8.5 LDA mutants. (A-O) DAPI staining (A-E) and neural patterning analysis of E8.5 embryos (9-12 somites) detects expression of NKX6.1 and PAX3 (red, green, respectively; F-J), and FOXA2 and SHH (red, green, respectively; K-O) in Ptch2−/−;Hhip1−/− (F,K), MT-Ptch1;Ptch1−/− (G,L), MT-Ptch1;Ptch1−/−;Ptch2−/− (H,M), MT-Ptch1;Ptch1−/−;Ptch2−/−;Hhip1+/− (I,N) and MT-Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− (J,O) embryos. Arrows indicate FOXA2 expression at the dorsalmost region of the neural tube (M,N). Scale bars: 50 μm. DEVELOPMENT 3430 RESEARCH ARTICLE Development 140 (16)

Fig. 6. Overlapping and distinct mechanisms of HH pathway antagonism by PTCH1, PTCH2 and HHIP1. (A) HH-responsive luciferase reporter activity measured from NIH/3T3 fibroblasts stimulated with constitutively active SmoM2 and co- transfected with the indicated constructs. Each condition was performed in triplicate and data are represented as mean±s.e.m. (n.s., not significant, P>0.05 two-tailed Student’s t-test). (B-E) HH-responsive luciferase reporter activity measured from Ptch1−/− mouse embryonic fibroblasts (MEFs) transfected with the indicated constructs. Data are expressed as luciferase reporter activity normalized to cells transfected with empty vector alone (pCIG) and represented as mean±s.e.m. Treatment with control- (white bars) or SHH-conditioned media (grey bars) is indicated in E. (F) COS7 cells were transfected with the indicated constructs and lysates were immunoprecipitated with anti- HA antibody and blotted with anti- GFP or anti-GAS1 antibodies. (G-O) Immunofluorescent detection of HA (green; G,J,M) and acetylated tubulin (ACTUB, red; H,K,N) in NIH/3T3 cells expressing PTCH1::HA (G-I), PTCH2::HA (J-L) and HHIP1::HA (M-O). Merged images with DAPI staining (blue) shown in I,L,O. Insets show ciliary localization of PTCH2::HA in Ptch1−/− MEFs (J-L). Scale bars: 5 μm.

et al., 1999; Taipale et al., 2002). To determine whether LIA by to SHH (Rahnama et al., 2004). We next determined whether PTCH2 involves a similar catalytic activity, we generated two PTCH2 could respond to SHH in Ptch1−/− MEFs. Interestingly, both analogous PTCH2 RND mutants: PTCH2G465V and PTCH1- and PTCH2-mediated inhibition of SMO is partially PTCH2D469Y. As previously reported, PTCH1G495V and relieved upon treatment with SHH (Fig. 6E), suggesting that PTCH1D499Y exhibit reduced ability to inhibit SMO in Ptch1−/− PTCH2 is responsive to SHH ligand. By contrast, a ligand- MEFs (Fig. 6C). Similarly, PTCH2G465V and PTCH2D469Y insensitive PTCH1 construct (PTCH1ΔL2) is refractory to SHH display impaired LIA at all concentrations tested (Fig. 6D), treatment (Fig. 6E). consistent with PTCH2 functioning as an RND permease. PTCH2 During vertebrate embryogenesis, the HH co-receptors GAS1, binds all three mammalian HH ligands with similar affinity as CDON and BOC are collectively required to initiate HH ligand- PTCH1 (Carpenter et al., 1998); however, previous work suggested mediated responses (Allen et al., 2011). Gas1;Cdon;Boc triple that PTCH2-mediated inhibition of SMO is only relieved after mutant embryos are nearly identical to Smo−/− mutants (Allen et al.,

treatment with desert hedgehog (DHH) ligand and does not respond 2011), yet HH signaling can be activated downstream of ligand DEVELOPMENT Inhibition of HH signaling RESEARCH ARTICLE 3431 using small-molecule SMO agonists in co-receptor deficient Similar to PTCH1, data presented here show that PTCH2 may cerebellar granule neuron precursors (Izzi et al., 2011). Consistent function as a receptor complex with the obligate HH co-receptors, with their role in mediating HH ligand-dependent signaling, GAS1, GAS1, CDON and BOC. This is consistent with the ability of CDON and BOC interact with PTCH1 and can form distinct PTCH2 to respond to SHH ligand, but future studies will be needed receptor complexes (Bae et al., 2011; Izzi et al., 2011). Based on to define the functional significance of these interactions and to the ability of PTCH2 to respond to SHH, we assessed whether determine how HHIP1 participates in the cell-surface HH PTCH2 interacts with the HH co-receptors by co- interactome. immunoprecipitation. Similar to PTCH1, HA-tagged PTCH2 PTCH1 is thought to function within the membrane of the interacts with GAS1, CDON and BOC in COS7 cells (Fig. 6F), primary cilium to prevent ciliary entry and subsequent activation of suggesting that PTCH2 can also form complexes with the HH co- SMO (Rohatgi et al., 2007). Consistent with this idea, we detect receptors. PTCH2 within primary cilium, implicating PTCH2 as a novel, SMO transduces the HH signal at the primary cilium, an ciliary-localized HH pathway component. However, the lack of organelle crucial for vertebrate HH signal transduction (Corbit et ciliary-localized HHIP1 suggests that ciliary localization is not a al., 2005; Huangfu et al., 2003). In the absence of ligand, PTCH1 general requirement for ligand-dependent HH pathway antagonism, localizes to the primary cilium to prevent SMO ciliary accumulation and that diverse mechanisms exist to restrict the activity of HH and activation. Ligand-binding to PTCH1 delocalizes LIA from the ligands during embryogenesis. ciliary membrane, enabling downstream signaling through SMO (Rohatgi et al., 2007). To determine whether PTCH2 and HHIP1 Collective requirement for PTCH2, HHIP1 and also localize to the primary cilium, we expressed HA-tagged PTCH1 during feedback antagonism of vertebrate PTCH2 and HHIP1 in NIH/3T3 cells to examine co-labeling of HA HH signaling with the ciliary marker, acetylated tubulin (ACTUB). Consistent Our data support a model where PTCH2, HHIP1 and PTCH1 with previous studies, PTCH1 localizes to the primary cilium comprise a semi-redundant feedback network of cell surface (Fig. 6G-I). Strikingly, we also detect PTCH2 within the ciliary antagonists that collectively act to restrict ligand-dependent HH membrane of transfected cells (Fig. 6J-L). This localization is not pathway activity (LDA). Removal of any single cell surface dependent on a physical interaction with endogenous PTCH1, as antagonist produces little to no defects in ventral cell specification, PTCH2::HA also localizes to the primary cilium in Ptch1−/− MEFs whereas combined removal of PTCH1-feedback inhibition and (Fig. 6J-L, insets). By contrast, HHIP1::HA does not localize to the either PTCH2 or HHIP1 produces a significant expansion of ventral primary cilium (Fig. 6M-O). Taken together, these data suggest that cell populations. Intriguingly, complete loss of feedback inhibition ciliary localization is a shared feature between PTCH1 and PTCH2, by all three cell surface antagonists yields a neural tube composed but that ciliary localization is not a universal requirement for ligand- entirely of ventral cell populations, including expression of the dependent HH pathway antagonism. highest-level HH targets, NKX2.2 and FOXA2, throughout the DV axis. DISCUSSION Patterning defects of this magnitude have thus far only been A novel role for PTCH2 as a HH pathway described for mutations that activate HH signaling downstream of antagonist during vertebrate neural patterning ligand, such as Ptch1−/− and Sufu−/− embryos (Cooper et al., 2005; Although initial studies suggested that PTCH2 plays little to no role Goodrich et al., 1997). By contrast, the severe ventralization in antagonizing HH signaling in vivo (Nieuwenhuis et al., 2006), observed in MT-Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− embryos likely data presented here support an important role for PTCH2 in results from loss of inhibition at the level of HH ligand (LDA) and restricting HH pathway activity during vertebrate embryogenesis. not simply from loss of SMO inhibition (LIA). First, only PTCH1 First, embryos lacking both PTCH1 and PTCH2 feedback inhibition and PTCH2 are capable of LIA; thus, the lack of ectopic signaling (MT-Ptch1;Ptch1−/−;Ptch2−/−) display more severe patterning in the dorsal neural tube of MT-Ptch1;Ptch1−/−;Ptch2−/− embryos defects than those lacking only PTCH1 feedback antagonism (MT- confirms that PTCH1 levels provided by the MT-Ptch1 transgene Ptch1;Ptch1−/−). This inhibitory role is most evident at E8.5, when are sufficient for LIA. As HHIP1 is incapable of LIA, the severe HH-dependent ventral patterning initially occurs unopposed by ventralization observed upon further removal of HHIP1 likely antagonistic roof plate signals, including Wnts and BMPs (Dudley results from enhanced ligand-dependent signaling. That HHIP1 is and Robertson, 1997; Parr et al., 1993). Second, the severe indirectly required for LIA is unlikely due to the normal patterning ventralization observed in MT-Ptch1;Ptch1−/−;Ptch2−/−;Hhip1−/− observed in Hhip1−/− and Ptch2−/−;Hhip1−/− embryos. Collectively, embryos compared with MT-Ptch1;Ptch1−/−;Hhip1−/− animals these results suggest an essential role for negative feedback at the suggests that PTCH2 limits HH activity in the absence of both level of HH ligand to restrict HH signaling during ventral neural HHIP1 and PTCH1 LDA. Last, chick electroporation studies and patterning. Of note, this mechanism is likely more global, as we cell signaling assays confirm that PTCH2 directly antagonizes HH also observe defects in somite patterning and craniofacial ligand function. This finding is particularly important in light of the development. potential role for PTCH2 in human cancers and congenital disorders Despite the overall similarities in patterning defects, there are (Fan et al., 2009; Fan et al., 2008). some clear differences between MT-Ptch1;Ptch1−/−;Ptch2−/−; We also present evidence that both PTCH1 and PTCH2 restrict Hhip1−/− (total LDA mutant) and Ptch1−/− (complete or near HH signaling using similar molecular mechanisms. Like PTCH1, complete LIA mutant) embryos. The floorplate, as demarcated by PTCH2 can antagonize ligand-dependent HH pathway activation the highest levels of FOXA2 and SHH synthesis, extends to the and directly inhibit SMO activity. Moreover, PTCH2-mediated LIA dorsal limits of the latter, but only to mid-regions of the neural tube depends on a conserved RND motif, implicating PTCH2 as a novel in the former. This may reflect the importance of timing in HH- RND permease within the HH pathway. Emerging evidence dependent patterning (Balaskas et al., 2012; Dessaud et al., 2008; suggests that the cell surface HH machinery functions in the context Dessaud et al., 2007). In Ptch1−/− embryos, ectopic signaling occurs

of a complex interaction network (Izzi et al., 2011; Bae et al., 2011). once cells gain competence to initiate a HH response. Conversely, DEVELOPMENT 3432 RESEARCH ARTICLE Development 140 (16) the ectopic pathway activity observed in complete LDA mutants is dependent on the kinetics of SHH ligand production and distribution. Dorsal progenitors in LDA mutants ultimately experience high levels of SHH, as indicated by FOXA2 and NKX2.2 expression; however, dorsal cells may not receive this signal within the narrow early competence window required for definitive floorplate specification (Ribes et al., 2010). Last, additional HH-binding proteins may limit the time and range of SHH ligand-based responses, including the HH co-receptors GAS1, CDON and BOC (Allen et al., 2011; Izzi et al., 2011), glypicans (Capurro et al., 2008; Li et al., 2011), megalin (LRP2) (Christ et al., 2012), or other cell-surface proteins.

Feedback regulation of SHH is required to establish discrete neural progenitor domain boundaries At the onset of ventral neural patterning, the SHH gradient induces or represses expression of transcriptional determinants along the DV axis to establish distinct progenitor fates. This initial pattern established by SHH ligand is thought to be inherently disorganized and must be refined by cross-repressive interactions between transcription factors expressed in neighboring domains, resulting in sharp boundaries between neural progenitor populations (Briscoe et al., 1999; Briscoe et al., 2000; Ericson et al., 1997). In fact, mathematical models of the downstream gene regulatory network (GRN) initiated by HH signaling can recapitulate the graded and discrete patterns established in the ventral neural tube independent of threshold responses to HH ligand (Balaskas et al., 2012). This suggests that precise interpretation of a SHH gradient is not required to establish distinct progenitor domains in the ventral neural tube (Balaskas et al., 2012). However, our observation of significant mixing of pV3 and pMN populations in embryos with compromised Fig. 7. Model of cell-surface regulation of HH signaling. In the absence of HH ligands (top panel), PTCH1 represses SMO activity (LIA). At LDA demonstrates the importance of feedback inhibition at the level the onset of HH signaling (middle panel), HH binding to PTCH1 and to of HH ligand to produce sharp boundaries between progenitor the obligate HH co-receptors GAS1, CDON and BOC results in de- populations and suggests that the downstream GRN is not sufficient repression of SMO function and initiation of a signal transduction cascade to properly pattern the ventral neural tube in the context of that culminates in GLI-mediated modulation of transcriptional targets. deregulated HH ligand. This initiates a negative-feedback mechanism at the cell surface that A recent study in zebrafish suggests that the initial noisy pattern includes the downregulation of Gas1, Cdon and Boc, and upregulation of established by HH ligand is corrected by dramatic cell Ptch1, Ptch2 and Hhip1. PTCH1, PTCH2 and HHIP1 binding to HH ligands rearrangements. These migratory events lead to clustering and (bottom panel) competes with productive ligand-receptor interactions to positioning of neural progenitors to establish discrete boundaries alter the balance between bound and unbound PTCH1, resulting in cell- between domains (Xiong et al., 2013). Strikingly, ectopic autonomous modulation of SMO activity. Additionally, ligand sequestration by cell-surface HH antagonists results in non-cell motoneurons induced in the zebrafish neural tube migrate into the autonomous HH pathway inhibition in cells distal to the HH source (LDA). appropriate region independent of their initial position (Xiong et al., 2013). Conservation of this mechanism in mice would predict that ectopic ventral progenitors in embryos with disrupted LDA should the unique role of Drosophila PTC has been distributed among three migrate into their appropriate positions and produce discrete partially redundant proteins in mammals (PTCH1, PTCH2 and boundaries. However, this is not the case, as our analysis reveals HHIP1), each of which are direct transcriptional targets of the SHH significant mixing of ectopic pMN and pV3 cells. This discrepancy pathway and each of which participates in direct LDA of SHH suggests that mouse and zebrafish could have fundamentally signaling (Fig. 7). The presence of additional antagonists may different mechanisms with which to achieve HH-dependent ventral provide essential robustness to HH-dependent patterning processes patterning. Alternatively, the ectopic progenitors in LDA mutants during vertebrate development, where HH ligands act over could have been specified after cells are epithelialized and are significantly larger distances and greater developmental times than therefore unable to migrate. One intriguing possibility is that the during invertebrate embryogenesis and in a broader variety of tissue proper regulation of HH ligands is required to direct these contexts. coordinated cell movements, despite an apparent lack of a role for Notably, the collective action of PTCH1, PTCH2 and HHIP1 to downstream signaling in this process (Xiong et al., 2013). restrict HH pathway activity is analogous to the general requirement for the HH co-receptors GAS1, CDON and BOC to activate HH Complexity of cell surface regulation of HH signaling (Allen et al., 2011; Izzi et al., 2011). Similarly, removal of signaling during vertebrate embryogenesis a single co-receptor produces only minor defects in ventral neural Together, the data presented here demonstrate that Drosophila and patterning while combined removal of GAS1, CDON and BOC

mammals have fundamentally similar feedback responses, though reveals their collective requirement in ligand-mediated HH pathway DEVELOPMENT Inhibition of HH signaling RESEARCH ARTICLE 3433 activation (Allen et al., 2011). The results presented in this study Capurro,M. I.,Xu,P.,Shi,W.,Li,F.,Jia,A.andFilmus,J.(2008). Glypican-3 define an equally important network of cell-surface antagonists that inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding. Dev. Cell 14, 700-711. are collectively required to antagonize ligand-dependent HH Carpenter, D., Stone, D. M., Brush, J., Ryan, A., Armanini, M., Frantz, G., signaling. However, it remains unclear what characteristics Rosenthal, A. and de Sauvage, F. J. (1998). Characterization of two patched distinguish GAS1, CDON and BOC as HH pathway activators receptors for the vertebrate hedgehog protein family. Proc. Natl. Acad. Sci. USA 95, 13630-13634. compared with the HH pathway antagonists examined in this study. Chen, Y. and Struhl, G. (1996). Dual roles for patched in sequestering and Future studies will be needed to elucidate the mechanisms that transducing Hedgehog. Cell 87, 553-563. regulate the balance between HH pathway activation and inhibition Chen, C. H., von Kessler, D. P., Park, W., Wang, B., Ma, Y. and Beachy, P. A. (1999). Nuclear trafficking of Cubitus interruptus in the transcriptional at the cell surface in different HH-responsive tissues during regulation of Hedgehog target gene expression. Cell 98, 305-316. embryogenesis, organ homeostasis and HH-dependent disease Christ, A., Christa, A., Kur, E., Lioubinski, O., Bachmann, S., Willnow, T. E. processes. and Hammes, A. (2012). LRP2 is an auxiliary SHH receptor required to condition the forebrain ventral midline for inductive signals. Dev. Cell 22, 268- Acknowledgements 278. We thank Dr M. P. Scott (Stanford University, CA, USA) for the MT-Ptch1 and Chuang, P. T. and McMahon, A. P. (1999). Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397, 617-621. Ptch1 mutant mice, and the Ptch1−/− MEFs. We also acknowledge Dr D. A. Chuang, P. T., Kawcak, T. and McMahon, A. P. (2003). Feedback control of Bumcrot (Curis, Lexington, MA, USA) for the Ptch2 mutant mice. We thank Dr mammalian Hedgehog signaling by the Hedgehog-binding protein, Hip1, Y. Nagakawa (University of Minnesota, Minneapolis, MN, USA) for the DBX1 modulates Fgf signaling during branching morphogenesis of the lung. Genes antibody. The NKX6.1, PAX3, FOXA2, NKX2.2, SHH, MNR2, ISL1, EVC1 and Dev. 17, 342-347. EN1 antibodies were obtained from the Developmental Studies Hybridoma Cooper, A. F., Yu, K. P., Brueckner, M., Brailey, L. L., Johnson, L., McGrath, J. Bank developed under the auspices of the NICHD and maintained by The M. and Bale, A. E. (2005). Cardiac and CNS defects in a mouse with targeted University of Iowa, Department of Biological Sciences, Iowa City, IA. Confocal disruption of suppressor of fused. Development 132, 4407-4417. microscopy was performed in the Microscopy and Image Analysis Laboratory Corbit, K. C., Aanstad, P., Singla, V., Norman, A. R., Stainier, D. Y. R. and at the University of Michigan. Reiter, J. F. (2005). Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018-1021. Funding Dessaud, E., Yang, L. L., Hill, K., Cox, B., Ulloa, F., Ribeiro, A., Mynett, A., Novitch, B. G. and Briscoe, J. (2007). Interpretation of the sonic hedgehog Work performed in F.C.’s lab is supported by grants from the Canadian morphogen gradient by a temporal adaptation mechanism. Nature 450, 717- Institute of Health Research (CIHR). F.C. is a Fonds de la recherche en santé du 720. Québec (FRSQ) Senior Scientist. A.M.H. was supported by the University of Dessaud, E., McMahon, A. P. and Briscoe, J. (2008). Pattern formation in the Michigan MSTP training grant [T32 GM007863] and the National Institutes of vertebrate neural tube: a sonic hedgehog morphogen-regulated Health (NIH) Cellular and Molecular Biology Training Grant [T32-GM007315]. transcriptional network. Development 135, 2489-2503. J.S. is supported by a research team grant through The University of Michigan Diez del Corral, R., Olivera-Martinez, I., Goriely, A., Gale, E., Maden, M. and Center For Organogenesis. This work was supported by an American Heart Storey, K. (2003). Opposing FGF and retinoid pathways control ventral neural Association scientist development grant [11SDG6380000] and by an NIH grant pattern, neuronal differentiation, and segmentation during body axis [5 R21 CA167122-02] to B.L.A. Deposited in PMC for release after 12 months. extension. Neuron 40, 65-79. Dudley, A. T. and Robertson, E. J. (1997). Overlapping expression domains of Competing interests statement bone morphogenetic protein family members potentially account for limited Dev. Dyn. The authors declare no competing financial interests. tissue defects in BMP7 deficient embryos. 208, 349-362. Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. M. and Briscoe, J. (1997). Pax6 controls progenitor Author contributions cell identity and neuronal fate in response to graded Shh signaling. Cell 90, Experiments were conceived and designed by A.M.H., A.P.M. and B.L.A., and 169-180. executed by A.M.H. J.Y.S. and A.M.H. performed the chick electroporations; Fan, Z., Li, J., Du, J., Zhang, H., Shen, Y., Wang, C.-Y. and Wang, S. (2008). A K.A.P. and Y.N. conducted the bioinformatic and transgenic analyses; S.M. and missense mutation in PTCH2 underlies dominantly inherited NBCCS in a F.C. designed and executed the protein interaction studies. A.M.H, A.P.M. and Chinese family. J. Med. Genet. 45, 303-308. B.L.A. wrote and edited the manuscript. Fan, Z., Du, J., Liu, H., Zhang, H., Dlugosz, A. A., Wang, C.-Y., Fan, M., Shen, Y. and Wang, S. (2009). A susceptibility locus on 1p32-1p34 for congenital Supplementary material macrostomia in a Chinese family and identification of a novel PTCH2 mutation. Supplementary material available online at Am. J. Med. Genet. A. 149A, 521-524. http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.095083/-/DC1 Freeman, M. (2000). Feedback control of intercellular signalling in development. Nature 408, 313-319. References Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A. and Scott, M. Allen, B. L., Song, J. Y., Izzi, L., Althaus, I. W., Kang, J.-S., Charron, F., Krauss, P. (1996). Conservation of the hedgehog/patched signaling pathway from flies Genes Dev. R. S. and McMahon, A. P. (2011). Overlapping roles and collective to mice: induction of a mouse patched gene by Hedgehog. 10, requirement for the coreceptors GAS1, CDO, and BOC in SHH pathway 301-312. function. Dev. Cell 20, 775-787. Goodrich, L. V., Milenković, L., Higgins, K. M. and Scott, M. P. (1997). Altered neural cell fates and medulloblastoma in mouse patched mutants. Science Bae, G.-U., Domené, S., Roessler, E., Schachter, K., Kang, J.-S., Muenke, M. 277, 1109-1113. and Krauss, R. S. (2011). Mutations in CDON, encoding a hedgehog receptor, Hooper, J. E. and Scott, M. P. (1989). The Drosophila patched gene encodes a result in holoprosencephaly and defective interactions with other hedgehog Cell Am. J. Hum. Genet. putative membrane protein required for segmental patterning. 59, 751- receptors. 89, 231-240. 765. Balaskas, N., Ribeiro, A., Panovska, J., Dessaud, E., Sasai, N., Page, K. M., Huangfu, D., Liu, A., Rakeman, A. S., Murcia, N. S., Niswander, L. and Briscoe, J. and Ribes, V. (2012). Gene regulatory logic for reading the Sonic Anderson, K. V. (2003). Hedgehog signalling in the mouse requires Cell Hedgehog signaling gradient in the vertebrate neural tube. 148, 273-284. intraflagellar transport proteins. Nature 426, 83-87. Briscoe, J., Sussel, L., Serup, P., Hartigan-O’Connor, D., Jessell, T. M., Hui, C. C. and Joyner, A. L. (1993). A mouse model of greig Rubenstein, J. L. and Ericson, J. (1999). Homeobox gene Nkx2.2 and cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an specification of neuronal identity by graded Sonic hedgehog signalling. intragenic deletion of the Gli3 gene. Nat. Genet. 3, 241-246. Nature 398, 622-627. Ingham, P. W., Nystedt, S., Nakano, Y., Brown, W., Stark, D., van den Heuvel, Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain M. and Taylor, A. M. (2000). Patched represses the Hedgehog signalling protein code specifies progenitor cell identity and neuronal fate in the ventral pathway by promoting modification of the Smoothened protein. Curr. Biol. 10, neural tube. Cell 101, 435-445. 1315-1318. Briscoe, J., Chen, Y., Jessell, T. M. and Struhl, G. (2001). A hedgehog- Izzi, L., Lévesque, M., Morin, S., Laniel, D., Wilkes, B. C., Mille, F., Krauss, R. insensitive form of patched provides evidence for direct long-range S., McMahon, A. P., Allen, B. L. and Charron, F. (2011). Boc and Gas1 each morphogen activity of sonic hedgehog in the neural tube. Mol. Cell 7, 1279- form distinct Shh receptor complexes with Ptch1 and are required for Shh-

1291. mediated cell proliferation. Dev. Cell 20, 788-801. DEVELOPMENT 3434 RESEARCH ARTICLE Development 140 (16)

Jeong, J. and McMahon, A. P. (2005). Growth and pattern of the mammalian Perrimon, N. and McMahon, A. P. (1999). Negative feedback mechanisms and neural tube are governed by partially overlapping feedback activities of the their roles during pattern formation. Cell 97, 13-16. hedgehog antagonists patched 1 and Hhip1. Development 132, 143-154. Peterson, K. A., Nishi, Y., Ma, W., Vedenko, A., Shokri, L., Zhang, X., Kawamura, S., Hervold, K., Ramirez-Weber, F.-A. and Kornberg, T. B. (2008). McFarlane, M., Baizabal, J.-M., Junker, J. P., van Oudenaarden, A. et al. Two patched protein subtypes and a conserved domain of group I proteins (2012). Neural-specific Sox2 input and differential Gli-binding affinity provide that regulates turnover. J. Biol. Chem. 283, 30964-30969. context and positional information in Shh-directed neural patterning. Genes Kutejova, E., Briscoe, J. and Kicheva, A. (2009). Temporal dynamics of Dev. 26, 2802-2816. patterning by morphogen gradients. Curr. Opin. Genet. Dev. 19, 315-322. Rahnama, F., Toftgård, R. and Zaphiropoulos, P. G. (2004). Distinct roles of Biochem. J. Lee, Y., Miller, H. L., Russell, H. R., Boyd, K., Curran, T. and McKinnon, P. J. PTCH2 splice variants in Hedgehog signalling. 378, 325-334. (2006). Patched2 modulates tumorigenesis in patched1 heterozygous mice. Ribes, V., Balaskas, N., Sasai, N., Cruz, C., Dessaud, E., Cayuso, J., Tozer, S., Cancer Res. 66, 6964-6971. Yang, L. L., Novitch, B., Martí, E. et al. (2010). Distinct Sonic Hedgehog signaling dynamics specify floor plate and ventral neuronal progenitors in the Li, F., Shi, W., Capurro, M. and Filmus, J. (2011). Glypican-5 stimulates vertebrate neural tube. Genes Dev. 24, 1186-1200. rhabdomyosarcoma cell proliferation by activating Hedgehog signaling. J. Cell Biol. Roelink, H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P. A. 192, 691-704. and Jessell, T. M. (1995). Floor plate and motor neuron induction by different McMahon, A. P., Ingham, P. W. and Tabin, C. J. (2003). Developmental roles Curr. Top. Dev. Biol. concentrations of the amino-terminal cleavage product of sonic hedgehog and clinical significance of hedgehog signaling. 53, 1-114. autoproteolysis. Cell 81, 445-455. Milenkovic, L., Goodrich, L. V., Higgins, K. M. and Scott, M. P. (1999). Mouse Rohatgi, R., Milenkovic, L. and Scott, M. P. (2007). Patched1 regulates Development patched1 controls body size determination and limb patterning. hedgehog signaling at the primary cilium. Science 317, 372-376. 126, 4431-4440. Sockanathan, S., Perlmann, T. and Jessell, T. M. (2003). Retinoid receptor Motoyama, J., Heng, H., Crackower, M. A., Takabatake, T., Takeshima, K., signaling in postmitotic motor neurons regulates rostrocaudal positional Tsui, L. C. and Hui, C. (1998a). Overlapping and non-overlapping Ptch2 identity and axonal projection pattern. Neuron 40, 97-111. expression with Shh during mouse embryogenesis. Mech. Dev. 78, 81-84. Taipale, J., Cooper, M. K., Maiti, T. and Beachy, P. A. (2002). Patched acts Motoyama, J., Takabatake, T., Takeshima, K. and Hui, C.-C. (1998b). Ptch2, a catalytically to suppress the activity of Smoothened. Nature 418, 892-896. second mouse Patched gene is co-expressed with Sonic hedgehog. Nat. Tseng, T. T., Gratwick, K. S., Kollman, J., Park, D., Nies, D. H., Goffeau, A. and Genet. 18, 104-106. Saier, M. H., Jr (1999). The RND permease superfamily: an ancient, ubiquitous Nakano, Y., Guerrero, I., Hidalgo, A., Taylor, A., Whittle, J. R. and Ingham, P. W. and diverse family that includes human disease and development proteins. J. (1989). A protein with several possible membrane-spanning domains encoded Mol. Microbiol. Biotechnol. 1, 107-125. by the Drosophila segment polarity gene patched. Nature 341, 508-513. Ulloa, F. and Briscoe, J. (2007). Morphogens and the control of cell proliferation Nieuwenhuis, E., Motoyama, J., Barnfield, P. C., Yoshikawa, Y., Zhang, X., and patterning in the spinal cord. Cell Cycle 6, 2640-2649. Mo, R., Crackower, M. A. and Hui, C.-C. (2006). Mice with a targeted Vokes, S. A., Ji, H., McCuine, S., Tenzen, T., Giles, S., Zhong, S., Longabaugh, mutation of patched2 are viable but develop alopecia and epidermal W. J. R., Davidson, E. H., Wong, W. H. and McMahon, A. P. (2007). Genomic hyperplasia. Mol. Cell. Biol. 26, 6609-6622. characterization of Gli-activator targets in sonic hedgehog-mediated neural Development Novitch, B. G., Wichterle, H., Jessell, T. M. and Sockanathan, S. (2003). A patterning. 134, 1977-1989. requirement for retinoic acid-mediated transcriptional activation in ventral Vokes, S. A., Ji, H., Wong, W. H. and McMahon, A. P. (2008). A genome-scale neural patterning and motor neuron specification. Neuron 40, 81-95. analysis of the cis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb. Genes Dev. 22, 2651-2663. Nybakken, K., Vokes, S. A., Lin, T.-Y., McMahon, A. P. and Perrimon, N. (2005). Wilkinson, D. G. (1992). In Situ Hybridization: A Practical Approach. Oxford, New A genome-wide RNA interference screen in Drosophila melanogaster cells for Nat. Genet. York: IRL Press at Oxford University Press. new components of the Hh signaling pathway. 37, 1323-1332. Xie, J., Murone, M., Luoh, S. M., Ryan, A., Gu, Q., Zhang, C., Bonifas, J. M., Okada, A., Charron, F., Morin, S., Shin, D. S., Wong, K., Fabre, P. J., Tessier- Lam, C. W., Hynes, M., Goddard, A. et al. (1998). Activating Smoothened Lavigne, M. and McConnell, S. K. (2006). Boc is a receptor for sonic mutations in sporadic basal-cell carcinoma. Nature 391, 90-92. Nature hedgehog in the guidance of commissural axons. 444, 369-373. Xiong, F., Tentner, A. R., Huang, P., Gelas, A., Mosaliganti, K. R., Souhait, L., Parr, B. A., Shea, M. J., Vassileva, G. and McMahon, A. P. (1993). Mouse Wnt Rannou, N., Swinburne, I. A., Obholzer, N. D., Cowgill, P. D. et al. (2013). genes exhibit discrete domains of expression in the early embryonic CNS and Specified neural progenitors sort to form sharp domains after noisy Shh limb buds. Development 119, 247-261. signaling. Cell 153, 550-561. DEVELOPMENT